In the present specification, tetrahydrobiopterin (L-erythro-5,6,7,8-tetrahydrobiopterin; hereinafter, referred to as THBP), 7,8-dihydrobiopterin (L-erythro-7,8-dihydrobiopterin; hereinafter, referred to as DHBP) or biopterin (hereinafter, referred to as BP) or a combination of any two or more of them is called a biopterin compound as a whole.
THBP is a biosubstance which is widely distributed in animals and was revealed to be a coenzyme of phenylalanine hydroxylase in liver of rat by Kaufman in 1963. After that, it was also revealed that THBP is a coenzyme which commonly acts on tyrosine hydroxylase and tryptophane hydroxylase and was revealed to play an important role participating in biosynthesis of neurotransmitters. In recent years, its function as a coenzyme for nitrogen monoxide (NO) synthase was also found and has been receiving public attention as a coenzyme for biosynthetic enzymes for various biosubstances. It has been known that, in human being, defect of THBP results in a reduction of activity of the above-mentioned amino acid hydroxylase causing an abnormally high phenylketonuria and abnormally high phenylalaninuria and THBP is used as a therapy for such inborn metabolism error diseases as sapropterin hydrochloride.
It has been known that, in animals, THBP is biosynthesized from guanosine triphosphate (hereinafter, abbreviated as GTP) by a three-step enzymatic reaction using GTP cyclohydrase I (hereinafter, abbreviated as GCH), 6-pyruvoyltetrahydropterin synthase (hereinafter, abbreviated as PTPS) and sepiapterin reductase (hereinafter, abbreviated as SPR) as shown in FIG. 1. In the meanwhile, other than the animals, THBP has been reported to be present in fruit fly (Weisberg, E. P. and O'Donnell, J. M. J. Biol. Chem. 261:1453-1458, 1996), silkworm (Iino, T., Sawada, H., Tsusue, M. and Takikawa, S.-I. Biochim. Biophys. Acta 1297:191-199, 1996), eukaryotic microbes such as Euglena gracillis, Neurospora crassa and Pycomyces blakesleeanus (Maier, J., Ninnemann, H. Photochamistry and Photobiology 61:43-53, 1995) and Nocardia species which are prokaryotic microbes (Son, J. K., Rosazza J. P. J. Bacteriol. 182:3644-3648, 2000). As such, although there are many living things in which the presence of THBP is reported, microbes are particularly greatly different from animals in view of evolution and, therefore, it is believed that THBP is not a biosubstance which is commonly available in microbes as a whole.
THBP is a compound having three asymmetric carbons in a molecular and, accordingly, it is a compound where its chemical synthesis is difficult and, with regard to its main synthetic methods, there has been known a method where L-erythro-biopterin (BP) is synthesized using rhamnose or deoxyarabinose as a starting material and then it is chemically subjected to an asymmetric reduction to synthesize the product. However, those chemically synthesizing methods have many reaction steps and, during the reaction steps, there are processes having low reaction yields. There are also difficulties that rhamnose and deoxyarabinose which are starting materials are expensive and they are hardly said to be advantageous manufacturing methods in terms of operation, yield, cost, and so on.
Under such circumstances, Kagamiyama, et al. tried the synthesis of THBP by an enzymatic reaction, purified each of the three enzymes participating in the THBP synthesis and by using the three enzymes succeeded in synthesizing the THBP in a reactor containing GTP and nicotinamide dinucleotide phosphate (reduced form) (hereinafter, abbreviated as NADPH) (Japanese Patent Laid-Open No. 82,888/1992). However, in obtaining 1 kg of THBP by this method, 3.12 kg of GTP and 92 kg of NADPH are necessary. When the facts that those materials are very expensive and that operations including purification of three kinds of enzymes are very troublesome are taken into consideration, industrial production of the biopterin compound using the said method is difficult in view of cost and operations.
On the other hand, Shiraishi, et al. found that enzymes of genus Candida (Candida noveiius, Candida rugosa, Candida robsta, etc.) and several filamentous fungi of genus Mucor (Mucor javanicus, Mucor alternns, Mucor subtilissmus, etc.) accumulate L-erythro-biopterin (BP) which is an oxidized product of THBP in a medium and reported a process for the production of BP from the culture medium utilizing the above property (Japanese Patent Laid-Open No. 9,296/1986 and No. 33,990/1993). Since BP is able to be converted to THBP by a chemical reduction, a method where BP is produced by microbes and then converted to THBP does not need expensive GTP and NADPH and, further, greatly reduces the synthesizing steps and, accordingly, it is an industrially advantageous manufacturing method in view of operations. However, in any of the strains, the amount of BP produced thereby is as low as that BP production per liter of the culture medium is not more than 1 mg and, therefore, it is not an industrially advantageous manufacturing method in view of yield and cost.
Accordingly, THBP which is useful as such has been hardly an object of study because there has been no industrially advantageous manufacturing method and, in addition, its usefulness has not been always sufficiently utilized because THBP has been hardly provided as pharmaceuticals, etc. in general. Further, although the biopterin compound such as DHBP and BP which are oxidized products of THBP which will be mentioned below have been expected to exhibit the same or even better pharmacological actions, the actions have not been fully investigated.